Reduced replication origin licensing selectively kills KRAS-mutant colorectal cancer cells via mitotic catastrophe

摘要

Western blot analysis shows that KRAS overexpression is at physiological levels and potent to induce downstream activation of ERK. Vinculin serves as a loading control. Quantification of KRAS protein expression after 1, 2, and 3 days of doxycycline induction. Mean expression ± SEM is shown (  = 2–3 per group). Representative growth curve of CaCo2 cells after KRAS induction shows no difference in growth with or without KRAS expression (  = 6 technical replicates; error bars = SEM). Graphical outline of the RNAi screen to identify genetic interactors of mutant KRAS. Change of shRNA abundance in the shRNA screen after 21 days of culture with and without doxycycline as determined by massive parallel sequencing. Each dot represents a shRNA. The -axis depicts the log fold change of shRNA abundance. The -axis shows each shRNA ranked by their fold change. The shRNA MCM7.1879 is specifically depleted when co-expressed with doxycycline induced KRAS . Western blots showing MCM7 expression after knockdown. KRAS and shRNA expression were induced with doxycycline for 0, 1, 3, 5, or 7 days. MCM7 knockdown was efficient after 3 days of shRNA expression. Vinculin serves as loading control. , Clonogenic assays of three non-overlapping shRNAs targeting MCM7 show increased sensitivity of KRAS expressing CaCo2 cells to MCM7 knockdown. Mean percentage of the relative area covered by cells ± SEM is shown (  = 3–4 per group). Student’s -test (two-sided); NS = not significant; *  p i Soft agar assays showing anchorage-independent growth in CaCo2 cells ± induced KRAS and ± MCM7.sh3. KRAS expression increases colony formation in CaCo2 cells about five fold. MCM7 knockdown has no detectable effect on cells expressing an empty control vector, whereas cells expressing additionally KRAS reduce colony formation significantly. Mean fold change of colonies formed after five weeks of culture is shown ± SEM (  = 3 per group). Student’s -test (two-sided); NS = not significant; ***  j Western blots show an increase in cleaved PARP (clPARP) staining after five and seven days of MCM7 knockdown specifically in CaCo2 cells expressing KRAS . ß-Tubulin and total PARP serve as a loading control, the cleaved PARP is indicated by an arrow. FACS analysis of cleaved Caspase 3 (clCaspase 3) reveals a significant increase of apoptosis in cells co-expressing KRAS and MCM7.sh3 compared to cells expressing MCM7.sh3 alone. Mean percentage of clCaspase 3 cells ± SEM is shown (  = 4 per group). Student’s -test (two-sided); ***  p